Electrophoresis in gels is a well-known procedure for determining the molecular weight of substances such as proteins, amino acids, nucleic acids, peptides and other macromolecules by applying an external electrical potential to a gel containing unresolved macromolecules in an electrophoresis cell and measuring the relative movements of the macromolecules. The molecular weight of a molecular specie can be calculated from a set of standards after obtaining a "Ferguson" plot to establish that the charge density of the unknown substance does not deviate from that exhibited by the standards.
The use of polyacrylamide gel electrophoresis (PAGE) allowed a separation effect based on a sieving effect imparted by control of the gel pore size in a "separating gel" layer, in addition to the separation obtained by electrophoretic mobility. However, molecular weight determination by PAGE was complicated by the wide range of electrophoretic charges possessed by the macromolecules present in the system. It was then discovered that these charge differences could be negated by the addition of sodium dodecyl sulfate (SDS) to the system. Large numbers of SDS molecules associate with each protein or macromolecule; the charge of the SDS molecules imparted to the SDS-macromolecule complex is so large that differences in charge, due to the composition of the macromolecule, are not detectable.
A particularly useful electrophoresis procedure is discontinuous SDS-PAGE developed in 1964 (U.S. Pat. No. 3,384,564). Discontinuous SDS-PAGE involves the use of a multi-phase (discontinuous) buffer system, varying in chemical composition, i.e., a systematic use of pH and buffer discontinuities.
Often in discontinuous SDS-PAGE, two separately polymerized layers of polyacrylamide, designated as a "stacking gel" and a "separating gel", are prepared. The stacking gel contains a low polymer concentration and has a relatively large pore size. The large pore size allows the sample to concentrate into tightly packed zones. The separating gel contains higher polymer concentrations and has a relatively small pore size. The polymer is the result of reaction between monomer and co-monomer or cross-linking agent. The effective pore size of the polymer is an inverse function of "total monomer concentration", percent T, defined as the sum of the concentrations of acrylamide and cross-linking agent. The small pore size provides a restrictive effect and produces resolution of the sample.
The uniqueness of this kind of discontinuous SDS-PAGE consists of its ability to concentrate the sample into a narrow starting zone necessary for good resolution. This is achieved in the stacking gel which differs in ionic composition and pH from the buffers in the electrode vessels.
Concentration of the sample into a narrow starting zone produces good resolution and occurs as an interface develops when the leading ion from the buffer of the stacking gel migrates out while the trailing ion of the electrolyte buffer replaces it, both moving in the same direction. The leading ion is chosen to have a higher effective mobility than the ionic species of the sample, and therefore migrates in front of all other ions. Behind the leading ion other zones form and concentrate. The concentrated macromolecule in the samples appear to the eye as one thin "stack". The concentrated sample "stack" continues to migrate through the stacking gel with no change in characteristics until it encounters a discontinuity in entering the separating gel, either in the nature of the supporting medium, i.e., pore size, or in the buffer, e.g., pH. This change produces the separation of the different macromolecular species into discrete bands. The overall procedure in discontinuous SDS-PAGE thus involves three stages: (a) stacking; (b) unstacking; and (c) resolution.
In practice, the gels are placed in a chamber containing buffer solutions, and the sample along with a tracking dye, is placed on top of the stacking gel and under the upper electrolyte buffer. After an electrical potential is applied, the sample is electrophoresed until the tracking dye migrates within a few mm from the bottom of the gel. The separated macromolecular bands are then stained for visualization. The tracking dye is used for the measurement of the relative mobility of these bands.
The true front is the boundary, which is the point of inflection of pH, between the leading and trailing ions. The point of inflection can be detected visually (as with the tracking dye), chemically, spectrometrically (refractive index) or by radioactive label. In the systems described herein, the leading ion of the buffer system is chloride ion. The point of inflection of a chloride-containing buffer system can be determined by precipitating and fixing the chloride ions with AgNO.sub.3. A more convenient method of measuring the true front comprises mixing an appropriate tracking dye with the sample to allow easy visual determination.
The tracking dye serves as the reference point for the measurement of the R.sub.f of the bands. The R.sub.f is defined as the distance that each band component has traveled from the top of the separating gel to the center of the band, divided by the distance that the leading front (tracking dye) has traveled. "Ferguson" plots of the log of R.sub.f versus the percent T (total monomer concentration of the separating gel) should be obtained to check for systematic errors. The molecular weight can be calculated by plotting a function of R.sub.f versus a function of molecular weight. While the R.sub.f values are often used for calculating the molecular weight of a macromolecular species, the R.sub.f values can be used in other ways, e.g., for determining the approximate comparative sizes of macromolecular species.
In a discontinuous SDS-PAGE system, the buffer and gel can be carefully controlled and reproduced. One of the major sources of error arises in measuring the R.sub.f. In order to provide the required accuracy and precision, the migration distances of the macromolecules must be accurate, and in a system containing a tracking dye this measurement depends upon the characteristics of the tracking dye. The tracking dye must migrate with the true front, i.e., at the interface of the leading and trailing ions. The present invention provides an improvement in tracking dyes suitable for use in discontinuous SDS-PAGE.